For the examination of biological specimens, it has been usual for some time to prepare the specimen with optical markers, in particular with fluorescent dyes. Often, for example in the field of genetic investigations, several different fluorescent dyes which become attached to specific specimen constituents are introduced into the specimen. From the fluorescent properties of the prepared specimen it is possible, for example, to draw conclusions as to the nature and composition of the specimen, or as to concentrations of specific substances within the specimen.
In scanning microscopy, a specimen is illuminated with a light beam in order to observe the detected light (in the form of reflected or fluorescent light) emitted by the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detected light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detection of the reflected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels via the beam deflection device back to the beam splitter, passes through the latter and is focused onto the detection pinhole, behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not originate directly from the focus region takes a different light path and does not pass through the detection pinhole, so that point like information is obtained which, by sequential scanning of the specimen with the focus of the illuminating light beam, results in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers. Commercial scanning microscopes usually comprise a scan module that is flange-mounted onto the stand of a conventional light microscope and contains all the aforesaid elements additionally necessary for scanning a specimen.
In confocal scanning microscopy, a detection pinhole can be dispensed with in the case of two-photon (or multi-photon) excitation, since the excitation probability depends on the square of the photon density and thus on the square of the illuminating light intensity, which of course is much greater at the focus than in the adjacent regions. The fluorescent light being detected therefore very probably originates almost exclusively from the focus region, which renders superfluous any further differentiation, using a pinhole arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.
For simultaneous illumination with light of several wavelengths, several lasers are usually used. EP 0 495 930 “Confocal microscope system for polychromatic fluorescence” discloses an arrangement having a single laser that emits several laser lines. In practice, mixed-gas lasers, in particular ArKr lasers, are used for this purpose. For detection, several detectors are usually provided for detected light of different wavelengths. One particularly flexible arrangement for simultaneous polychromatic detection of detected light of several wavelengths is disclosed in German Patent DE 199 02 625 “Apparatus for simultaneous detection of several spectral regions of a laser beam.”
Many fluorescent dyes can be excited only with ultraviolet illuminating light. The use of ultraviolet illuminating light has the disadvantage, especially for living specimens, of much more severe specimen damage. In addition, all optical components must be transparent to ultraviolet light and to the fluorescent light, which because of Stokes shifting has a longer wavelength, and must not be damaged by illumination with ultraviolet light. With cemented optical components in particular, such as lens element groups in a microscope objective, illumination with ultraviolet light results in irreversible damage to the cement and the lens elements. A further disadvantage of illumination with ultraviolet light arises from its shallower penetration depth into biological specimens. The disadvantages can be eliminated using two-photon or multi-photon excitation. In multi-photon scanning microscopy, the fluorescent photons attributable to a two-photon or multi-photon excitation process are detected. The probability of a two-photon transition depends on the square of the excitation light power level. In order to achieve high light power levels, it is therefore advantageous to pulse the illuminating light in order to achieve high peak pulse power levels. This technique is known, and is disclosed e.g. in U.S. Pat. No. 5,034,613 “Two-photon laser microscopy” and in the German Patent Application DE 44 14 940. A further advantage of multi-photon excitation, in confocal scanning microscopy in particular, is improved bleaching characteristics, since the specimen bleaches out only in the region of sufficient power density, i.e. at the focus of an illuminating light beam. In contrast to single-photon excitation, almost no bleaching takes place outside this region.
All known methods and arrangements for examination of a specimen based on multi-photon excitation are limited to excitation of a single laser line. For many applications, it is essential to mark the specimen specifically with different fluorescent dyes or other markers, in order to obtain information concerning the three-dimensional structure or composition of the specimen by simultaneous excitation of all the fluorescent dyes and polychromatic detection. Simultaneous excitation of several UV fluorescent dyes is at present possible only with single-photon excitation, so that the negative effects of irradiation with ultraviolet light are fully evident.